Native Acylated homoserine lactone diffusion tests

We perfomed simple tests in liquid culture with different AHL concentrations and saw different fluorescent intensities. We also did diffusion experiments on Agar to characterize the diffusion speed and distance depending on the concentrations. Those datas are used for the Model of the AHL diffusion.The concentrations used in the experiments were based on the paper handling the evaluation of a focused library of AHL and previous results with the plate reader experiment below.

On the one hand double layer Agar diffusion was tested which was not always succesfull. Double layer Agar consists of a first traditional 1.5% Agar layer. The specificity of the double layer Agar is the second 0.7% Agar layer containing receiver cells. In the experiments the AHL drop was placed in the middle and the diffusion was observed over everal hours. The concentration we tested were [10 uM];[100 uM] and [1 mM]

For the single layer Agar diffusion (the more realistic model and adapted to the applications for the GAME BOARD) we place receiver colonies on the Agar in a spiral pattern(Figure 1.2 and Figure 2.2) to avoid a "shadow" behind the cells. An AHL drop of 2 uL was placed in on the central colony an the diffusion was observed over several hours.
After 5 hours of incubation we can interpret two things from images and grey values. First of all the background noise is relatively high and the two highest concentrations diffuse at the same distance until 5 hours of incubation. The lowest concentration (10 uM) diffuse less far than the higher concentrations, confirmed by the images and grey value analysis.</p>

Figure 1.1 Evolution of Grey value over relative colony distance after 5 hours.The expectation of what we see under 365nm wavelength illumination is digitally comfirmed using the grey values for the different colonies

Figure 1.2 AHL diffusion on agar plate after 5hours.Agar plate containing receiver cell colonies in spiral pattern. Image taken 23 hours after adding the drop of AHL on the central colony. Concentration :[10 uM] (top left);[100 uM](top right) and [1 mM](Bottom left) as well as a negative control(Bottom right). On the left side an image at 365 nm wavelength, on the right side the same picture in greyscale

After 23 hours of incubation the difference is clearly visible in the 365 nm exposure images as well as from grey values. As expected, depending on the [AHL],the GFP expression in different colonies are activated more or less far away from origin (central colony).The background noise (due to leacky expression ) is stil relatively high bvut the triggered GFP expression is clearly visible, espacially in the grey scale 365nm wavelenght images.

Figure 2.1 Evolution of Grey value over relative colony distance after 23 hours. The expectation of what we see under 365nm wavelength illumination is digitally comfirmed using the grey values for the different colonies

Figure 2.2 AHL diffusion on agar plate after 23hours. Agar plate containing receiver cell colonies in spiral pattern. AHL drop (2 uL) was added on the central colony. Concentration :[10 uM] (top left);[100 uM](top right) and [1 mM](Bottom left) as well as a negative control(Bottom right). On the left side an image at 365 nm wavelength, on the right side the same picture in greyscale

Native Acylated homoserine lactone tests in liquid culture using the plate reader Tecan infinite M2000 PRO

File:Gfp od sigmoid1.png

I order to select mutated pLuxR promoters we need to know about the sensivity of the Wild-Type type. The test range was inspired form the paper about Evaluation of a focused libraryu of N-Acryl L-Homoserine lactone reveals a new set of potent quorum sensing modulators.The paper shows sensivities of the pLuxR promoter to different sets of AHL molecules in Vibrio fisheri. We need to readjuste the ranges for our E.coli DH5alpha strain in a second run and finally get our sensivity curve over the linear range of [0 nM],[0.25 nM],[0.5 nM],[1 nM],[2 nM],[3 nM],[4 nM],[5 nM],[10nM],[20 nM],[30 nM],[40 nM]and[50 nM],.

The experimental set-up included a 96-well plate filled with 180 uL LB media , 10uL of receiver cells and 10 uL of different AHL concentrations, everything in triplicates. (+ blank and negative control). The experiment runs for 16 hours in order to monitor the evolution of fluorescence and later choose the steady state to create the sensivity curve.The datas are analyzed with GraphPad Prism 6.0.